Characterization of PC12 Cell Subclones with Different Sensitivities to Programmed Thermal Stimulation.

Neuritogenesis is the process underling nervous system regeneration; however, optimal extracellular signals that can promote neuronal regenerative activities require further investigation. Previously, we developed a novel method for inducing neuronal differentiation in rat PC12 cells using temperature-controlled repeated thermal stimulation (TRTS) with a heating plate. Based on neurogenic sensitivity to TRTS, PC12 cells were classified as either hyper- or hyposensitive. In this study, we aimed to investigate the mechanism of hyposensitivity by establishing two PC12-derived subclones according to TRTS sensitivity during differentiation: PC12-P1F1, a hypersensitive subclone, and PC12-P1D10, a hyposensitive subclone. To characterize these subclones, cell size and neuritogenesis were evaluated in subclones treated with nerve growth factor (NGF), bone morphogenetic protein (BMP), or various TRTS. No significant differences in cell size were observed among the parental cells and subclones. BMP4- or TRTS-induced neuritogenesis was increased in PC12-P1F1 cells compared to that in the parental cells, while no neuritogenesis was observed in PC12-P1D10 cells. In contrast, NGF-induced neuritogenesis was observed in all three cell lines. Furthermore, a BMP inhibitor, LDN-193189, considerably inhibited TRTS-induced neuritogenesis. These results suggest that the BMP pathway might be required for TRTS-induced neuritogenesis, demonstrating the useful aspects of these novel subclones for TRTS research.

Sciatic nerve leachate of cattle causes neuronal differentiation of PC12 cells via ERK1/2 signaling pathway.

Previous studies have shown that the sciatic nerve has neurotrophic activity, and nerve regeneration, differentiation, and axon outgrowth can be modulated by different sciatic nerve preparations. However, numerous animals may have to be sacrificed to obtain enough sciatic nerves to make a sciatic nerve preparation. Some studies have demonstrated that the role of sciatic nerve preparations in neural differentiation depends on the neurotrophins that Schwann cells secrete, and these factors are highly conserved among different species. To reduce the use of experimental animals, in this study, we made a leachate by using the sciatic nerve of cattle and explored its effect on neuronal differentiation of rat PC12 cells (a useful model for studying neuronal differentiation). Results showed the neurite outgrowth of PC12 cells treated with the cattle sciatic nerve leachate for 3, 6, and 9 days was significantly improved, and the expressions of ß3-tubulin and microtubule-associated protein 2 (two neuron-specific proteins) were increased. Moreover, the ERK1/2 signaling pathway was activated after PC12 cells were incubated with cattle sciatic nerve leachate for 9 days. Thus, a sciatic nerve leachate obtained from cattle can effectively induce neuronal differentiation of rat PC12 cells via ERK1/2 signaling pathway.

Secretome of Differentiated PC12 Cells Restores the Monocrotophos-Induced Damages in Human Mesenchymal Stem Cells and SHSY-5Y Cells: Role of Autophagy and Mitochondrial Dynamics.

A perturbed cellular homeostasis is a key factor associated with xenobiotic exposure resulting in various ailments. The local cellular microenvironment enriched with secretory components aids in cell-cell communication that restores this homeostasis. Deciphering the underlying mechanism behind this restorative potential of secretome could serve as a possible solution to many health hazards. We, therefore, explored the protective efficacy of the secretome of differentiated PC12 cells with emphasis on induction of autophagy and mitochondrial biogenesis. Monocrotophos (MCP), a widely used neurotoxic organophosphate, was used as the test compound at sublethal concentration. The conditioned medium (CM) of differentiated PC12 cells comprising of their secretome restored the cell viability, oxidative stress and apoptotic cell death in MCP-challenged human mesenchymal stem cells and SHSY-5Y, a human neuroblastoma cell line. Delving further to identify the underlying mechanism of this restorative effect we observed a marked increase in the expression of autophagy markers LC3, Beclin-1, Atg5 and Atg7. Exposure to autophagy inhibitor, 3-methyladenine, led to a reduced expression of these markers with a concomitant increase in the expression of pro-apoptotic caspase-3. Besides that, the increased mitochondrial fission in MCP-exposed cells was balanced with increased fusion in the presence of CM facilitated by AMPK/SIRT1/PGC-1α signaling cascade. Mitochondrial dysfunctions are strongly associated with autophagy activation and as per our findings, cellular secretome too induces autophagy. Therefore, connecting these three potential apices can be a major breakthrough in repair and rescue of xenobiotic-damaged tissues and cells.

Coenzyme Q10 attenuated ß-amyloid25-35-induced inflammatory responses in PC12 cells through regulation of the NF-κB signaling pathway.

Inflammation plays critical roles in the pathogenic mechanisms of several neurodegenerative disorders including Alzheimer's disease (AD). Previous study revealed that CoQ10 augmented cellular antioxidant defense capacity, thereby protecting PC12 cells from oxidative neurotoxicity. However, the mechanism by which CoQ10 inhibits inflammation remains unknown. In this study, we aim to examine the effects of CoQ10 on Aß25-35-induced inflammatory in PC12 cells and the underlying molecular mechanism of its neuroprotective action. CoQ10 suppressed the protein expression of COX-2 and the level of PGE2 in Aß25-35-injured PC12 cells. These inhibitions appeared to correlate with the suppression of NF-κB activation by CoQ10, as pretreating PC12 cells with CoQ10 blocked the translocation of NF-κB into the nuclear compartment and degradation of the inhibitory subunit IκB. Overall, these results implied that CoQ10 attenuated neuroinflammatory responses through the inactivation of NF-κB dependent inflammatory pathways in Aß25-35-induced PC12 cells. Therefore, CoQ10 may have therapeutic potential for neurodegenerative diseases by inhibiting pro-inflammatory mediators production.

Nanomechanical and topographical imaging of living cells by atomic force microscopy with colloidal probes.

Atomic Force Microscopy (AFM) has a great potential as a tool to characterize mechanical and morphological properties of living cells; these properties have been shown to correlate with cells' fate and patho-physiological state in view of the development of novel early-diagnostic strategies. Although several reports have described experimental and technical approaches for the characterization of cellular elasticity by means of AFM, a robust and commonly accepted methodology is still lacking. Here, we show that micrometric spherical probes (also known as colloidal probes) are well suited for performing a combined topographic and mechanical analysis of living cells, with spatial resolution suitable for a complete and accurate mapping of cell morphological and elastic properties, and superior reliability and accuracy in the mechanical measurements with respect to conventional and widely used sharp AFM tips. We address a number of issues concerning the nanomechanical analysis, including the applicability of contact mechanical models and the impact of a constrained contact geometry on the measured Young's modulus (the finite-thickness effect). We have tested our protocol by imaging living PC12 and MDA-MB-231 cells, in order to demonstrate the importance of the correction of the finite-thickness effect and the change in Young's modulus induced by the action of a cytoskeleton-targeting drug.

Hypergravity stimulation enhances PC12 neuron-like cell differentiation.

Altered gravity is a strong physical cue able to elicit different cellular responses, representing a largely uninvestigated opportunity for tissue engineering/regenerative medicine applications. Our recent studies have shown that both proliferation and differentiation of C2C12 skeletal muscle cells can be enhanced by hypergravity treatment; given these results, PC12 neuron-like cells were chosen to test the hypothesis that hypergravity stimulation might also affect the behavior of neuronal cells, in particular promoting an enhanced differentiated phenotype. PC12 cells were thus cultured under differentiating conditions for either 12 h or 72 h before being stimulated with different values of hypergravity (50 g and 150 g). Effects of hypergravity were evaluated at transcriptional level 1 h and 48 h after the stimulation, and at protein level 48 h from hypergravity exposure, to assess its influence on neurite development over increasing differentiation times. PC12 differentiation resulted strongly affected by the hypergravity treatments; in particular, neurite length was significantly enhanced after exposure to high acceleration values. The achieved results suggest that hypergravity might induce a faster and higher neuronal differentiation and encourage further investigations on the potential of hypergravity in the preparation of cellular constructs for regenerative medicine and tissue engineering purposes.

Nanogrooved surface-patterns induce cellular organization and axonal outgrowth in neuron-like PC12-cells.

Modulation of a materials surface topography can be used to steer various aspects of adherent cell behaviour, such as cell directional organization. Especially nanometric sized topographies, featuring sizes similar to for instance the axons of the spiral ganglion cells, are interesting for such purpose. Here, we utilized nanosized grooves in the range of 75-500 nm, depth of 30-150 nm, and pitches between 150 nm and 1000 nm for cell culture of neuron-like PC12 cells. The organizational behaviour was evaluated after 7 days of culture by bright field and scanning electron microscopy. Nanotopographies were shown to induce aligned cell-body/axon orientation and an increased axonal outgrowth. Our findings suggest that a threshold for cell body alignment of neuronal cells exists on grooved topographies with a groove width of 130 nm, depth of 70 nm and pitch of 300 nm, while axon alignment can already be induced by grooves with 135 nm width, 52 nm depth and 200 nm pitch. However, no threshold has been found for axonal outgrowth, as all of the used patterns increased outgrowth of PC12-axons. In conclusion, surface nanopatterns have the potential to be utilized as an electrode modification for a stronger separation of cells, and can be used to direct cells towards the electrode contacts of cochlear implants.

Imaging the post-fusion release and capture of a vesicle membrane protein.

The molecular mechanism responsible for capturing, sorting and retrieving vesicle membrane proteins following triggered exocytosis is not understood. Here we image the post-fusion release and then capture of a vesicle membrane protein, the vesicular acetylcholine transporter, from single vesicles in living neuroendocrine cells. We combine these measurements with super-resolution interferometric photo-activation localization microscopy and electron microscopy, and modelling to map the nanometer-scale topography and architecture of the structures responsible for the transporter's capture following exocytosis. We show that after exocytosis, the transporter rapidly diffuses into the plasma membrane, but most travels only a short distance before it is locally captured over a dense network of membrane-resident clathrin-coated structures. We propose that the extreme density of these structures acts as a short-range diffusion trap. They quickly sequester diffusing vesicle material and limit its spread across the membrane. This system could provide a means for clathrin-mediated endocytosis to quickly recycle vesicle proteins in highly excitable cells.

Involvement of GPR12 in the induction of neurite outgrowth in PC12 cells.

GPR12, an orphan G protein-coupled receptor, constitutively activates the Gs signaling pathway and further increases intracellular cyclic AMP. GPR12 overexpression has been reported to promote neurite extension in neurons or transform neuro2a neuroblastoma cells into neuron-like cells. However, the possible effects and mechanisms of GPR12 in the differentiation of PC12 cells are still unknown. The present study shows that GPR12 overexpression induced PC12 cells differentiation into neuron-like cells with enlarged cell sizes and neuritogenesis possibly via activation of Erk1/2 signaling and significantly increased the expression of several neurite outgrowth-related genes, including Bcl-xL, Bcl-2 and synaptophysin. These findings indicate that GPR12 may play a role in neurite outgrowth during PC12 cell differentiation.

Huperzine A derivative M3 protects PC12 cells against sodium nitroprusside-induced apoptosis.

AIM: To investigate the effects of M3, a derivative of huperzine A, on the apoptosis induced by sodium nitroprusside (SNP) in PC12 cells. METHODS: Cell viability was detected using MTT method. Apoptosis was examined with annexin V/prodium iodide (PI) stain. The levels of reactive oxygen species (ROS) were measured using fluorophotometric quantitation. The amount of malonaldehyde (MDA) was determined with MDA detection kits. The expression of caspase-3 and Hsp70 were analyzed using Western blotting. RESULTS: Exposure of PC12 cells to SNP (200 µmol/L) for 24 h decreased the cell viability to 69.0% of that in the control group. Pretreatment with M3 (10 µmol/L) or huperzine A (10 µmol/L) significantly protected the cells against SNP-induced injury and apoptosis; the ratio of apoptotic bodies in PC12 cells was decreased from 27.3% to 15.0%. Pretreatment with M3 (10 µmol/L) significantly decreased ROS and MDA levels, and increased the expression of Hsp70 in the cells. Quercetin (10 µmol/L) blocked the protective effect of M3, while did not influence on that of huperzine A. CONCLUSION: M3 protects PC12 cells against SNP-induced apoptosis, possible due to ROS scavenging and Hsp70 induction.

Oxidative mechanisms contribute to nanosize silican dioxide-induced developmental neurotoxicity in PC12 cells.

Neurotoxicity was investigated in nano-SiO2-treated cultured PC12 cells, an in vitro neuronal cell model, in order to define a relatively safe dose range for its application. The following were observed in the present study: (1) A dose-dependent increase in the level of reactive oxygen species (ROS) with a corresponding decrease in the level of glutathione (R2=0.965) suggesting 20- and 50-nm SiO2-induced free radical generation and glutathione depletion. (2) A dose- and time-dependent decrease in cell viability that was associated with elevation of ROS level, especially after 24-h nano-SiO2 exposure (R2=0.965), suggesting the role of oxidative stress on nano-SiO2 induced cell death. (3) An increase in the level of thiobarbituric-acid reactive species that correlated reversely with cell viability of the PC12 cells treated with nano-SiO2 (R2=0.945) suggesting nano-SiO2-induced membrane damage caused by lipid peroxidation. (4) A dose-dependent increase in sub-G1 population in SiO2-exposed cells along with cell shrinkage and nuclear condensation from morphological examination suggesting nano-SiO2-induced cell apoptosis. Furthermore, nano-SiO2 exposure diminished the ability of neurite extension in response to nerve growth factor in treated PC12 cells. In summary, SiO2 nanoparticle exposure resulted in dose-dependent neurotoxicity in cultured PC12 cells that was probably associated with oxidative stress and induced apoptosis.

Characterization of Dp71Δ(78-79), a novel dystrophin mutant that stimulates PC12 cell differentiation.

Dp71 has an important role in the central nervous system. To better understand the function of Dp71 domains in neuronal differentiation, PC12 cells were stably transfected with a dystrophin mutant, Dp71Δ(78-79) , which lacks exons 78 and 79. Based on the percentage of cells bearing neurites and neurite length analyses, we found that cells stably expressing Dp71Δ(78-79) (PC12-C11) differentiate more efficiently than non-transfected cells. While wild-type cells reach their maximum differentiation 9-12 days after initiating the differentiation process, the PC12-C11 cells reach differentiation in 4-6 days. Protein expression analysis showed a down-regulation of Dp71a and an up-regulation of Dp71ab and/or Up71, ß-dystroglycan and neuron-specific enolase in undifferentiated and in neural growth factor differentiated PC12-C11 cells. No change was observed in the expression of Grb2 and Up400. The subcellular localization of Dp71Δ(78-79) was in the cell periphery, and there was no change in localization during the differentiation process, which was also observed throughout the neurite extensions.

6-Hydroxydopamine-induced PC12 cell death is mediated by MEF2D down-regulation.

Recently, it was reported that in a 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model, neuronal cell death is associated with the cdk5-mediated hyperphosphorylation of myocyte enhancer factor 2 (MEF2), a transcription factor that is critically required for neuronal survival. In the present study, we investigated the possible involvement of cdk5-mediated MEF2D down-regulation on 6-hydroxydopamine (6-OHDA)-induced PC12 cell death. 6-OHDA was found to significantly increase nitric oxide (NO) production and to induce apoptosis in a time-dependent manner in PC12 cells. Furthermore, 6-OHDA was found to markedly reduce MEF2D levels under conditions that could induce PC12 cell apoptosis. In addition, PC12 cell death and MEF2D degradation by 6-OHDA were prevented by the cdk5 inhibitor roscovitine, but roscovitine could not restore the 6-OHDA-induced inactivation of Akt. These results suggest that the cell death and MEF2D degradation caused by 6-OHDA are dependent on cdk5 activity. On the other hand, roscovitine enhanced the 6-OHDA-induced activations of ERK1/2 and JNK, but reduced the 6-OHDA-induced activation of p38. These results suggest that PC12 cell death by 6-OHDA appears to be regulated by the down-regulation of MEF2D via some interaction between cdk5 and MAP kinase.

Aucubin modulates Bcl-2 family proteins expression and inhibits caspases cascade in H2O2-induced PC12 cells.

In this study, the effect of aucubin on H(2)O(2)-induced apoptosis was studied by using a rat pheochromocytoma (PC12) cell line. We have analyzed the apoptosis of H(2)O(2)-induced PC12 cells, H(2)O(2)-induced apoptosis appeared to correlate with lower Bcl-2 expression, higher Bax expression and sequential activation of caspase-3 leading to cleavage of poly-ADP-ribose polymerase (PARP). Aucubin not only inhibited lower Bcl-2 expression, high Bax expression, but also modulated caspase-3 activation, PARP cleavage, and eventually protected against H(2)O(2)-induced apoptosis. These results indicated that aucubin can obstruct H(2)O(2)-induced apoptosis by regulating of the expression of Bcl-2 and Bax, as well as suppression of caspases cascade activation.

Genistein attenuates D-galactose-induced oxidative damage through decreased reactive oxygen species and NF-κB binding activity in neuronal PC12 cells.

AIMS: We investigated the mechanism of D-galactose (DG)-induced oxidative damage and the neuroprotective action of genistein in PC12 cells. MAIN METHODS: PC12 cells were treated with 40mM DG dissolved in medium containing 85% RPMI1640, 10% HBS and 5% FBS with or without genistein. We measured the protein expression of ß-amyloid (Aß), advanced glycation end products (AGEs), IκB-α and manganese-superoxide dismutase (MnSOD) by western blotting, intracellular reactive oxygen species (ROS) by 2, 7-dichlorofluorescin-diacetate, and the binding activity of nuclear factor kappa B (NF-κB) by electrophortic mobility shift assay. KEY FINDINGS: DG (40mM) completely retarded cell growth after incubation for 72h, and this effect was not due to osmotic changes, as 40mM mannitol had no effect. Mechanistically, we found that DG increased intracellular ROS starting at 4h and increased Aß and AGEs at 24h. DG treatment for 24h also increased the binding activity of NF-κB but strongly decreased the expression of IκB-α protein. Furthermore, DG treatment for 48h increased MnSOD protein expression. All these effects of DG were effectively inhibited by genistein (0.5-10µM). SIGNIFICANCE: The present study indicates that the protection of genistein against DG-induced oxidative stress in PC12 cells, and the effect is likely mediated by decreased intracellular ROS and binding activity of NF-κB.

Taurine protection of PC12 cells against endoplasmic reticulum stress induced by oxidative stress.

BACKGROUND: Taurine is a free amino acid present in high concentrations in a variety of organs of mammalians. As an antioxidant, taurine has been found to protect cells against oxidative stress, but the underlying mechanism is still unclear. METHODS: In this report, we present evidence to support the conclusion that taurine exerts a protective function against endoplasmic reticulum (ER) stress induced by H2O2 in PC 12 cells. Oxidative stress was introduced by exposure of PC 12 cells to 250 uM H2O2 for 4 hours. RESULTS: It was found that the cell viability of PC 12 cells decreased with an increase of H2O2 concentration ranging from approximately 76% cell viability at 100 uM H2O2 down to 18% at 500 uM H2O2. At 250 uM H2O2, cell viability was restored to 80% by taurine at 25 mM. Furthermore, H2O2 treatment also caused a marked reduction in the expression of Bcl-2 while no significant change of Bax was observed. Treatment with taurine restored the reduced expression of Bcl-2 close to the control level without any obvious effect on Bax. Furthermore, taurine was also found to suppress up-regulation of GRP78, GADD153/CHOP and Bim induced by H2O2, suggesting that taurine may also exert a protective function against oxidative stress by reducing the ER stress. CONCLUSION: In summary, taurine was shown to protect PC12 cells against oxidative stress induced by H2O2. ER stress was induced by oxidative stress and can be suppressed by taurine.

Diazoxide preconditioning alleviates caspase-dependent and caspase-independent apoptosis induced by anoxia-reoxygenation of PC12 cells.

Although there is increasing evidence that the ATP sensitive potassium channel (K(ATP)) opener exhibits neuroprotective effects against ischaemic neural damage, little is known about the mechanism. Mitochondria play a key role in apoptosis by releasing many important factors, including cytochrome c and apoptosis-inducing factor, which in turn initiate the caspase-dependent and -independent mitochondrial pathway, respectively. In the present study, we sought to determine the locus that K(ATP) opener uses to mediate this protection in PC12 cells. We found that pre-treatment of PC12 cells with diazoxide (DZX), a mitochondrial ATP sensitive potassium channel (mitoK(ATP)) opener, dose-dependently increased cell viability under conditions of oxygen glucose deprivation (OGD). The protective effect of this pre-conditioning was attenuated by 5-hydroxydecanoic acid, a selective mitoK(ATP) blocker. The results showed that DZX inhibits the release of cytochrome c, the activation of caspase-3 and the release of AIF evoked by OGD. Taken together, our results demonstrate for the first time that activation of the mitoK(ATP) channel elicits protective effects against OGD-induced cell apoptosis by caspase-dependent and -independent mitochondrial pathways.

Up-regulation of Idh3alpha causes reduction of neuronal differentiation in PC12 cells.

The PC12 is the widely used cell line to study neuronal differentiation. We had extensively investigated the details of protein expression in differentiated PC12 cells by proteomic analysis. The cells were incubated at the presence of nerve growth factor. We had analyzed the expression changes in the differentiating PC12 cells by 2-dimensional electrophoresis and the identification of the proteins using MALDI-TOF MS. By comparing expression pattern in the time course, we identified the candidate genes which are associated with neuronal differentiation. Among these genes, we performed real-time PCR analysis to validate Idh3alpha expression by the time course. To identify the function of Idh3alpha in neuronal differentiation stage, the transfection of Idh3alpha to PC12 cells was performed. As a result, we proved that up-regulation of Idh3alpha causes reduction in neural differentiation of PC12 cells. Based on these data, we suggest that Idh3alpha plays a role to the neuronal differentiation.

Recreational drugs, 3,4-Methylenedioxymethamphetamine(MDMA), 3,4-methylenedioxyamphetamine (MDA) and diphenylprolinol, inhibit neurite outgrowth in PC12 cells.

3,4-Methylenedioxymethamphetamine (MDMA) is widely abused as a psychoactive recreational drug. It is well known that MDMA induces neurotoxic damage of serotonergic nerve endings. Although drug abuse is increasing among youths, it is unclear whether recreational drugs affect the development of nerve growth. Thus, the present study examined the effect of recreational drugs, such as MDMA, 3,4-methylenedioxyamphetamine (MDA) and diphenylprolinol, a novel recreational drug with a similar chemical structure as that of psychoactive agent pipradrol, on nerve growth factor (NGF)-induced neurite outgrowth. These recreational drugs induced a dose-dependent cell death in PC12 cells. The IC(50) values of MDMA, MDA, R-diphenylprolinol and S-diphenylprolinol were 4.11 mM, 2.75 mM, 1.00 mM and 0.77 mM, respectively, at 24 hr. To examine the effects of these recreational drugs on NGF-induced neurite outgrowth, PC12 cells were treated with NGF together with MDMA, MDA, S-diphenylprolinol or R-diphenylprolinol at low toxic concentrations. The recreational drugs significantly suppressed neurite outgrowth of PC12 cells induced by NGF. The results suggest that these psychoactive recreational drugs may inhibit neurite growth and thus be implicated in their elicited neurotoxicity.

Nerve growth factor-induced differentiation of PC12 cells is accompanied by elevated adenylyl cyclase activity.

Rat pheochromocytoma (PC12) cells characteristically undergo differentiation when cultured with nerve growth factor (NGF). Here we show that NGF dramatically increased the adenylyl cyclase-activating property of forskolin in PC12 cells. This effect of NGF was well maintained even when NGF was removed after 4 days, even though the morphological features of neuronal differentiation were rapidly lost on removal of NGF. The enhanced cAMP production in response to forskolin could be due to a synergistic interaction between forskolin and endogenously released agonists acting on G(s)-coupled receptors. However, responses to forskolin were not attenuated by antagonists of adenosine A2 receptors or pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, suggesting that adenosine and PACAP were not involved. Adenylyl cyclases 3, 6 and 9 were the predominant isoforms expressed in PC12 cells, but we found no evidence for NGF-induced changes in expression levels of any of the 9 adenylyl cyclase isoforms, nor in the expression of Gα(s). These findings highlight that NGF has a subtle influence on adenylyl cyclase activity in PC12 cells which may influence more than the neurite extension process classically associated with neuronal differentiation.